The use of prodrugs represents a clinically very valuable concept in cancer therapy since, particularly where the prodrug is to be converted to an anti-tumour agent under the influence of an enzyme that is linkable to a monoclonal antibody that will bind to a tumour associated antigen, the combination of such a prodrug with such an enzyme monoclonal/antibody conjugate represents a very powerful clinical agent. This approach to cancer therapy, often referred to as "antibody directed enzyme/prodrug therapy" (ADEPT) is disclosed in WO88/07378.
A further therapeutic approach termed "virus-directed enzyme prodrug therapy" (VDEPT) has been proposed as a method for treating tumour cells in patients using prodrugs. Tumour cells are targeted with a viral vector carrying a gene encoding an enzyme capable of activating a prodrug. The gene may be transcriptionally regulated by tissue specific promoter or enhancer sequences. The viral vector enters tumour cells and expresses the enzyme, in order that a prodrug is converted to an active drug within the tumour cells (Huber et al, Proc. Natl. Acad. Sci. USA (1991) 88, 8039). Alternatively, non-viral methods for the delivery of genes have been used. Such methods include calcium phosphate co-precipitation, microinjection, liposomes, direct DNA uptake, and receptor-mediated DNA transfer. These are reviewed in Morgan & French, Annu. Rev. Biochem., 1993,62; 191. The term "GDEPT" (gene-directed enzyme prodrug therapy) is used to include both viral and non-viral delivery systems.
Naturally-occurring enediyne antibiotics, including dynemicin Konishi, M. et al, J. Chem. Soc. 1990, 112, 3715-3716, esperamicin (Golik, J. et al, J. Amer. Chem. Soc. 1987, 109, 3462-3464) and calicheamicin (Lee, M. D. et al, J. Amer. Chem. Soc. 1987, 109, 3464-3466) are very potent cytotoxins, with IC.sub.50 values for inhibition of growth of tumour cell cultures in the low pM range. This extreme potency makes them attractive as potential effectors for prodrugs (Maier, M. E. Synlett. 1995, 13-26). The cytotoxic effects of these compounds are triggered by molecular rearrangements which bring the conjugated triple bonds of the enediyne core sufficiently close to initiate an electrocyclic reaction (Bergman, R. G. Acc. Chem. Res. 1973, 6, 25-31). Formation of a transient benzene 1,4-diradical capable of simultaneously abstracting a proton (at C-4' or C5') from a ribose moiety on each DNA chain results in a cascade of radical reactions leading to generation of double strand breaks (De Voss, J. J. et al, J. Amer. Chem. Soc. 1990, 112, 9669-9670).
While the natural antibiotics are very complex molecules, recent work (Nicolaou, K. C. et al, Science 1992, 256, 1172-1178; Nicolaou, K. C. et al, Proc. Natl. Acad. Sci USA. 1993, 90, 5881-5888) has described a series of simpler and more accessible synthetic analogues that are also very potent cytotoxins. A variety of releasing systems, including base-catalysed .beta.-elimination and and photolytically generated systems have been reported (Nicolaou, K. C., et al J. Amer. Chem. Soc. 1992, 114, 8890-8907: Nicolaou, K. C., et al, J. Amer. Chem. Soc. 1993, 115, 7944-7953: Nicolaou, K. C.; Dai, W.-M. J. Amer. Chem. Soc. 1992, 114, 8908-8921: Wender, P. A. et al, J. Org. Chem. 1993, 58, 5867-5869: Wender P. A. et al, Synthesis, 1994, 1279-1282).
4-Nitrobenzyloxycarbonyl derivatives of a variety of classes of cytotoxins have been tested for their ability to be substrates for the aerobic nitroreductase enzyme isolated from E. coli. Following reduction of the nitro group of the 4-nitrobenzyloxycarbonyl group by nitroreductase enzymes, the group is destabilised and released from the cytotoxin. 4-Nitrobenzyloxycarbonyl compounds proposed in the art include derivatives of phenylenediamine mustard, actinomycin D, mitomycin C, and doxorubicin. The degree of activation of these compounds by the enzyme varies widely and unpredictably (Anlezark, G. M. et al, J. Biochem. Pharmacol. 1992, 44, 2289-2295: Knox, R. J. et al, Cancer Metastasis Rev. 1993, 12, 195-212: Knox, R. J. et al, Biochem. Pharmacol. 1992, 44, 2297-2301: Mauger, A. B. et al, J. Med. Chem. 1994, 37, 3452-3458).
We have now found that 4-nitrobenzylcarbamate derivatives of enediynes are unexpectedly good substrates for nitroreductase, and are thus of potential use as prodrugs for GDEPT in conjunction with this enzyme.